DOI: http://dx.doi.org/10.22034/ari.2016.107493

Archives of Razi Institute, Vol. 72, No. 1 (2017) 23-31 Copyright © 2017 by Razi Vaccine & Serum Research Institute

Original Article Effects of experimental Mesobuthus eupeus envenomation on chicken

Khosravi ∗∗∗ 1, M., Mayahi 2 , M., Jalali 2 , S.M., Rezaie 1 , A., Taghavi Moghadam 3 , A., Hosseini 4 , Z., Barzegar 4 , S.K., Azadmanesh 4,S.

1. Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Khuzestan, Iran 2. Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Khuzestan, Iran 3. Razi Reference Laboratory of Scorpion Research, Razi Vaccine and Serum Research Institute, Ahvaz, Iran 4. Student of Veterinary Medicine, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Khuzestan, Iran

Received 13 January 2016; accepted 24 April 2016 Corresponding Author: [email protected]

ABSTRACT This study aimed to evaluate the clinical, histopathological and hematological effects of Mesobuthus eupeus venom on chicken organs. Adult chickens were subcutaneously injected with five doses of M. eupeus venom (0.5, 2, 5, 10 and 20 mg/kg; four chickens per each dose). Symptoms were recorded during the experiment and blood samples were collected for hematological analysis. Moreover, a complete necropsy was performed. After macroscopic examination, tissue samples were obtained from the liver, kidneys, heart, lungs, intestines and brain of the chickens three days after venom administration. In intravenous injection, lethal dose of the venom was determined at 15 mg/kg. The first clinical, pathological and hematological symptoms in envenomated chickens were observed at M. eupeus doses of 2, 5 and 0.5 mg/kg, respectively. Hematological examination revealed a reduction in lymphocyte count following experimental envenomation, which returned to the pre-experiment level in almost all the cases. On the other hand, heterophil count was found to increase during the experimental period. In addition, erythrocyte count and hematocrit level were stable at all the intervals. Pathological examination was indicative of severe pulmonary hemorrhage, pulmonary and cerebral edema, tubular necrosis of the kidneys, hemorrhage in kidneys and heart, hyaline thrombus and congestion of the liver. According to the results of this study, poultry are resistant to the toxic effects of M. eupeus venom. Keywords: Mesobuthus eupeus , Venom, Chicken, Pathology, Hematology

Les effets d’une envenimation expérimentale au scorpion Mesobuthuseupeus chez le poulet Résumé: Cette étude avait pour but d’évaluer les effets cliniques, histopathologiques et hématologiques du venin de Mesobuthus eupeus sur les organes du poulet.Cinq doses de venin de M. eupeus (0,5, 2, 5, 10 and 20 mg/kg) ont été injectées par voie sous-cutanée aux poulets (chaque dose injectée à un groupe de 4 poulets). Les symptômes ont été enregistrés au cours de chaque expérience et des prélèvements sanguins ont été soumis à des analyses hématologiques. De plus, une nécropsie complète a été menée. Après examen macroscopique, des prélèvements du foie, des reins, du cœur, des poumons, des intestins et cerveau ont été effectués trois jours après l’administration du venin. La dose létale de venin par injection intraveineuse a été déterminée à 15 mg/kg. Les premiers symptômes cliniques, pathologiques et hématologiques d’envenimation au M. eupeus ont été respectivement observés à des doses de venins de 2, 5 et 0,5 mg/kg. Les examens histopathologiques ont révélés

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une réduction des lymphocytes juste après l’envenimation avec un retour aux taux pré-expérimentaux dans presque tous les cas analysés. D’une autre part, une augmentation dans la numération d’hétérophile a été observée alors que les taux d’érythrocytes et d’hématocrites restaient stables aux différents intervalles analysés. L’examen pathologique indiquait de sévères hémorragies pulmonaires, des œdèmes pulmonaires et cérébraux, une nécrose tubulaire des reins, des hémorragies rénales et cardiaques, des thromboses hyalines et une congestion du foie. Selon nos résultats, les volailles montrent une résistance aux effets toxiques du venin de M. eupeus. Mots clés: Mesobuthus eupeus , Venin, Poulet, Pathologie, Hématologie

INTRODUCTION and symptoms may manifest within a few minutes or are a major health hazard for humans and days after the sting. Evaluation of the history, , especially in tropical regions (Bawaskar and symptoms, haematological and chemical factors are Bawaskar, 2012; Warrell, 2012). Scorpions belong to essential to the accurate diagnosis of envenomation and the Phylum Arthropoda , class Arachnida and order prediction of the status of victims. Previous studies in Scorpiones . Mesobuthus eupeus species belong to the this regard have denoted various biochemical family, which are responsible for the majority (Radmanesh, 1990; Taghavi Moghdam et al., 2009), of envenomation cases in the Middle East and central hematological (Emam et al., 2008; Taghavi Moghdam Asia, particularly in Iran (Karatas, 2003; Sadeghian, et al., 2009) and pathological manifestations (Kumar et 2003; Dehghani and Khamehchian, 2008). Several al., 2012; Zayerzadeh et al., 2012) to be induced by toxic fractions in M. eupeus venom may give rise to scorpion envenomation in humans, rabbits, mice and sting symptoms (Tuuri and Reynolds, 2011; Sagheb et rats. Median lethal dose of different scorpion venoms al., 2012). As short-chain peptides, biological ranges between 0.25-3.6 mg/kg in injections to mice. In compounds (Adiguzel, 2010) and bioactive substances the majority of lethal scorpions, LD50 is below 1.5 in scorpion venom (e.g., enzymes, peptides, mg/kg (Karatas, 2003). Vulnerability of different nucleotides, lipids, mucoproteins, biogenic amines and animals to the toxic effects of scorpion envenomation is other unknown compounds) (Boyer et al., 2009) could variable (Dehesa-Dávila and Possani, 1994; Padilla et affect vertebrate and invertebrate organisms (Upadhyay al., 2003). Therefore, assessment of the level of and Ahmad, 2008). Out of an estimated amount of resistance to various toxins in animals could result in 100,000 distinct peptides that exist in scorpion venom, the recognition of the action mechanisms of toxic approximately 400 peptides have been shown to exert compounds. Considering the differences between birds toxic effects on humans and animals (Karatas, 2003). and mammal species and lack of sufficient data Massive release of catecholamines following scorpion regarding the effects of scorpion venom on poultry envenomation (Gueron et al., 1993) influences various organs, this study aimed to evaluate the clinical, regulatory hormones, such as glucagon, cortisol and histopathological and hematological effects of angiotensin (Radha et al., 1998). Stimulation of the Mesobuthus eupeus venom on chicken organs. sympathetic system leads to adrenergic stimulation, MATERIALS AND METHODS which is associated with cardiac, metabolic, respiratory and neuromuscular disturbance (Chippaux, 2012). Venom preparation. In this study, Mesobuthus Consequences of scorpion envenomation mainly eupeus scorpions were collected from different regions depend on the species of scorpions, venom compounds of Khuzestan province in the southwest of Iran (31°19 ′- and physiological response of the victim. These signs 32°73 ′ N, 48°41 ′-49°4 ′ E) and milked by electric

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stimulation at the end of the tail. Freeze-dried venom performed immediately afterwards. Blood smears were was dissolved in distilled water and dialyzed against prepared on glass slides. After fixation, prepared distilled water at the temperature of 4 °C for 48 hours. samples were stained with Giemsa solution for After dialysis, venom solution was centrifuged at 1500 differential leukocyte counting. In the next step, we rpm for 15 minutes, and the supernatant was collected. determined total leukocytes, lymphocytes, heterophils, On the day of envenomation, crude venom was diluted erythrocytes and hematocrit level. Furthermore, total with distilled water to obtain the final protein counts of erythrocytes and leukocytes were recorded concentrations of 0.5, 2, 5, 10 and 20 mg/kg per body through manual haemocytometer chamber counting. In weight of the chickens. Total protein concentration was total, 100 lymphocytes, neutrophils, monocytes, measured using regular Bradford spectrophotometry eosinophils, and basophils were counted in order to with standard bovine serum albumin. determine the relative microscopic differential counts. Animals. In this study, 24 adult broiler chickens Moreover, hematocrit level was verified through blood were equally divided into six groups (n=4), categorized centrifugation in a capillary tube. as A-F. Subjects in the control group (A) received 500 Pathological analysis. A complete necropsy was L of ultra-pure water via subcutaneous injection into carried out on two randomly selected animals from the breast region using a disposable 1-mL hypodermic each experimental group, including those that died or syringe. Via an identical route, animals in the had to be euthanized three days after venom experimental groups received 500 L of a solution administration. Macroscopic examination was containing 500 g (group B), 2 mg (group C), 5 mg performed, and tissue samples were obtained from the (group D), 10 mg (group E), and 20 mg (group F) of liver, kidneys, heart, lungs, intestines and brain of the the scorpion venom dissolved in ultra-pure water. At 48 animals. Tissue processing and staining were hours after venom injection, symptoms were recorded conducted using conventional methods, as previously in all the study groups. described (Bancroft and Gamble, 2007). In brief, organ Toxicity verification. All the experiments in this samples of the chickens with venom injection were study were performed in accordance with the ethical immersed in 10% formalin for fixation. After guidelines of the National Ethics Advisory Committee dehydration with ethanol and clarification with xylene, (2006). To determine toxicity, higher concentrations of tissue sections of the organs were embedded in the scorpion venom (50-200 µg) were administered via paraffin. In the next stage, 5-m sections were prepared subcutaneous (SC) and intraperitoneal injection (IP) to from the embedded tissues. Paraffin sections were albino mice (weight: 20±2 g). Moreover, increased placed on glass slides, dewaxed with xylene and concentrations of the venom (5-40 mg/kg) were rehydrated with distilled water. Afterwards, sections administered via intravenous (IV) and SC injection to were dried and stained with haematoxylin and eosin. adult chickens. Following the treatment, animals were Finally, a minimum of two well-prepared slides monitored for 24 hours, and the number of dead containing each tissue sample were further studied animals was recorded at the end of the experiment. In using a light microscope. addition, lethal dose of the scorpion venom was Statistical analysis. In this study, statistical calculated at this stage. significance of the differences between the study Hematological analysis. At intervals of 0.5, 1, 2, 4, groups was assessed using one-way analysis of 6 and 24 hours after venom administration, venous variance (ANOVA) and Tukey’s post-hoc test. In blood samples were collected from study groups (A, B, all statistical analyses, P value of less than 0.05 C, E and F) using 10% ethylenediamine tetra-acetic was considered significant. acid anticoagulant, and hematological evaluation was

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RESULTS Clinical presentation. In this study, no specific clinical symptoms were observed in groups A (control) and B (0.5 mg/kg venom injection) at different time intervals. On the other hand, shortness of breath and lethargy were observed in the chickens administered with 2 mg/kg of the scorpion venom (group C) after one hour. It is noteworthy that the clinical signs diminished after two hours, and complete symptom remission occurred after four hours. Clinical symptoms of envenomation manifested 20 minutes after the SC administration of 5 and 10 Figure 1. The effect of experimental M. eupeus mg/kg of scorpion venom in groups D and E, envenomation on heterophil counts of chickens (A: control, B: 0.5, C: 2, E: 10, F: 20 mg/kg). respectively. These symptoms included increased respiration, increased oral secretion, and bloody stool. Complete symptom remission in these groups occurred after 24 hours. However, in study group F, additional symptoms of envenomation prevailed until the end of the experimental period (e.g., breathing problems, excessive mucus secretion, diarrhea, and leg paralysis). Furthermore, SC administration of 30 mg/kg of scorpion venom immediately caused mucous diarrhea, lameness, increased respiration and drooping wings in the animals. After one hour, clinical signs of envenomation were recorded as increased heart rate and respiratory rate, breathing disorders, excessive mucus secretion, Figure 2. The effect of experimental M. eupeus diarrhea, leg paralysis, neck rotation, seizures, and envenomation on lymphocyte counts of chickens (A: death (after two hours). However, experimental mice control, B: 0.5, C: 2, E: 10, F: 20 mg/kg). died following the SC injection of 11.5 mg/kg of the venom. According to our findings, lethal doses of M. eupeus venom in IV administration were 15 and 4.5 mg/kg in chickens and mice, respectively. Hematological findings. In hematological examinations, maximal reduction of lymphocyte count was recorded in all the study groups six hours after the treatment (figures 1, 2 & 3). Although changes in this regard were not statistically significant, total leukocyte count was observed to elevate significantly after 24 hours in experimental group F (P=0.018). Moreover, heterophil count was observed to increase in all the study groups Figure 3. The effect of experimental M. eupeus envenomation on total leukocyte counts of chickens (A: two hours after venom injection, and this change was control, B: 0.5, C: 2, E: 10, F: 20 mg/kg).

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considered significant in group F after 24 hours chickens were 15 and 30 mg/kg in IV and SC (P=0.039). Our findings were indicative of no administration, respectively; these values have been significant changes in the erythrocyte count and reported by previous studies conducted on mice hematocrit level at different time intervals. However, a (Khoobdel et al., 2013). slight reduction was observed in all the study groups in this regard. Accordingly, lymphocyte count returned to pre-injection levels in all the groups after 24 hours, while the heterophil count constantly increased during the experiment. Pathological signs. No pathological manifestations were observed in study groups A, B and C (Figure 4). On the other hand, pathological examination revealed different lesions in study groups D, E and F (figures 5, 6 & 7), including severe pulmonary hemorrhage and pulmonary edema, which were characterized by the accumulation of hyaline degeneration around the vessels and bronchi. Moreover, cardiac hemorrhage, tubular necrosis, congestion in the kidneys, hyaline Figure 5. Photomicrograph of effects of experimental M. thrombus, and congestion of the liver were observed in eupeus envenomation on study group D (H&E staining; A. lungs, pulmonary edema (arrows); B. heart, normal structure; the animals. The mentioned symptoms appeared to be C. liver, accumulation of hyaline degeneration in sinusoids; D. dose -dependent, and the most severe lesions were kidneys, congestion (arrows) and tubular necrosis (stars) reported in study group F.

Figure 6. Photomicrograph of effects of experimental M. Figure 4. Photomicrograph of effects of experimental M. eupeus envenomation on study group E (H&E staining; A. eupeus envenomation on study group A (H&E staining; note lungs, pulmonary edema (arrows); B. heart, haemorrhage (star); to normal structure of A. lungs, B. heart, C. liver and D. C. liver, accumulation of hyaline degeneration in sinusoids kidneys) (arrows); D. kidneys, congestion (arrows) and tubular degeneration (stars)

DISCUSSION This finding is suggestive of the higher resistance of According to the results of the present study, chickens to the toxic effects of M. eupeus scorpion approximate lethal doses of M. eupeus venom in venom compared to mice. In the current research, the

28 Khosravi et al / Archives of Razi Institute, Vol. 72, No. 1 (2017) 23-31 first clinical, pathological and hematological symptoms According to the results of the present study, total of envenomation in chickens were observed at doses of count of heterophils in animals of experimental groups 2, 5 and 0.5 mg/kg, respectively. was higher compared to the control group, with the maximum value observed in chickens administered with the highest dose of M. eupeus venom (group F).

Table 1. Hematological values for chickens (Wakenell, 2010) Parameter Interval Leukocytes (/µl) 12,000-30,000 Heterophils 3000-6,000 Lymphocytes 7,000-17,500 Packed cell 22-35 volume (%) Erythrocytes (/µl) 2,500,000-3,500,000

This is in line with the hematological signs reported in other species. Neutrophilic leukocytosis has been previously reported in humans (Gueron and Ovsyshcher, 1987; Gueron et al., 1993; Bucaretchi et al., 1995) and animals with scorpion envenomation Figure 7. Photomicrograph of effects of experimental M. eupeus envenomation on study group F (H&E staining; A. (Cordeiro et al., 2006; Ribeiro et al., 2009; Pinto et al., lungs, pulmonary edema (arrows); B. heart, haemorrhage (star); 2010). This condition is considered a consequence of C. liver, accumulation of hyaline degeneration in sinusoids (arrows); D. kidneys, congestion (arrows) and tubular necrosis stress and recruitment of heterophils to circulating (stars) blood compartments. According to the results of the The symptoms of scorpion envenomation in humans current research, erythrocyte count and hematocrit level are excessive sweating, severe pain in the site of sting, were stable in envenomated chickens. This finding is in increased salivation and vomiting, generalized tingling congruence with the results of a study conducted on and numbness, transient hypertension, tachycardia, Wistar rats with envenomation (Pinto et al., 2010), tachypnoea, and pulmonary edema (Natu et al., 2006). while inconsistent with another investigation on These symptoms are similar to those of envenomated envenomated dogs (Ribeiro et al., 2009). In the present chickens and reported signs in other animal species. In study, lymphocyte values were observed to decrease in humans, manifestations of the central nervous system envenomated chicken, which was previously reported following scorpion envenomation start with abnormal in envenomated dogs (Cordeiro et al., 2006; Nogueira behaviors, such as altered sensorium, agitation, et al., 2007). However, different results have been confusion and delirium (Das et al., 1995). In a study in proposed in dogs and rats with envenomation (Ribeiro this regard, cerebrovascular manifestations with et al., 2009; Pinto et al., 2010). After scorpion neurological deficits were reported in four patients out envenomation, the highest concentrations of toxins are of 50 cases with scorpion envenomation (Upadhyay found in the kidneys, liver, heart and lungs and Ahmad, 2008). Similar neurological symptoms (Petricevich, 2010). Furthermore, these organs are were observed in the chickens with scorpion highly susceptible to the pathological effects of envenomation in the present study, which is indicative scorpion venom in chicken. Scorpion venom contains of the same mechanism of toxic peptides in poultry variable concentrations of neurotoxins, cardiotoxins, and mammals. Normal hematological values for nephrotoxins, phosphodiesterases, hyaluronidases, chickens are presented in Table 1 (Wakenell, 2010). glycosaminoglycans, histamine, serotonin, tryptophans,

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and cytokine-releasing peptides (Mahadevan, 2000). According to the results of this study, M. eupeus These toxins may cause diverse pathological venom affects the organs of broiler chicken similar to manifestations. Previous studies have focused on the other animal species. Furthermore, review of the pathological effects of scorpion envenomation in literature is indicative of the higher resistance of humans and experimental animals (e.g., rabbits, rats poultry to the toxic effects of scorpion venom and mice). Moreover, some of the pathohistological compared to human. complications caused by the inflammatory cytokine induction of the M. eupeus venom include severe Ethics alveolar edema and hemorrhage, thrombosis, I hereby declare all ethical standards have been congestion, interstitial lung inflammation, respected in preparation of the submitted article. myocytolysis, coagulative necrosis, myocardial edema, and cardiac hemorrhage (Zayerzadeh et al., 2012). Conflict of Interest Consistently, these manifestations were observed in the The authors declare that they have no conflict of interest. chicken organs evaluated in the present study.In a Grant Suport research in this regard, cause of death in two individuals with envenomation was reported to be This study was financially supported by a grant from pulmonary edema, which is in line with the results of Shahid Chamran University of Ahvaz, Iran. the current research (Das et al., 2013). Pulmonary Acknowledgments edema may occur due to cardiac failure, hemodynamic disorders or release of chemical mediators (Kumar et Hereby, we extend our gratitude to the personnel of al., 2012). Furthermore, cardiorespiratory failure Razi Vaccine and Serum Research Institute for through the release of catecholamines and myocardial assisting us in this research project. ischemia are other causes of death in cases with envenomation (Karnad, 1998; Cupo and Hering, 2002; References Maheshwari and Tanwar, 2012). In-vivo effects of M. Adiguzel, S., 2010. In vivo and in vitro effects of scorpion eupeus venom have been previously investigated in venoms in Turkey: a mini-review. Journal of Venomous mice, rats and rabbits as alternative animal models Animals and Toxins including Tropical Diseases 16, 198- 211. (Khamechian et al., 2009; Lowe, 2010). In the current Bawaskar, H.S., Bawaskar, P.H., 2012. Scorpion sting: study, we evaluated the in-vivo effects of M. eupeus update. J Assoc Physicians India 60, 46-55. venom on chicken organs based on hematological and Boyer, L.V., Theodorou, A.A., Berg, R.A., Mallie, J., histological analyses. According to the results, the main Arizona Envenomation, I., Chavez-Mendez, A., et al., organs targeted by M. eupeus venom are the liver, 2009. Antivenom for critically ill children with kidneys, lungs and brain; however, the most severe neurotoxicity from scorpion stings. N Engl J Med 360, 2090-2098. damages caused by envenomation occurred in the Bucaretchi, F., Baracat, E.C., Nogueira, R.J., Chaves, A., respiratory and nervous systems. This is inconsistent Zambrone, F.A., Fonseca, M.R., et al., 1995. A with previous findings in this regard, which suggested comparative study of severe scorpion envenomation in kidneys, liver and spleen as the major susceptible children caused by bahiensis and . organs to envenomation by H. lepturus or venom of Rev Inst Med Trop Sao Paulo 37, 331-336. other scorpions belonging to the Buthidae family. This Chippaux, J.P., 2012. Emerging options for the management discrepancy could be due to the use of different of scorpion stings. Drug Des Devel Ther 6, 165-173. Cordeiro, F.F., Sakate, M., Fernandes, V., Cuyumjian, P.R., experimental animals and venom dosages. 2006. Clinical and cardiovascular alterations produced by scorpion envenomation in dogs. Journal of Venomous

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